Dendritic cell-based immunotherapy for cancer and relevant challenges for transfusion medicine.

Dendritic Cell–Based Immunotherapy for Cancer and Relevant Challenges for Transfusion Medicine

Ching Y.Voss,Mark R.Albertini,and James S.Malter

The encouraging results from dendritic cell–related can-cer immunotherapy have created tremendous interest for its broad clinical application.Dendritic cells are the most potent antigen-presenting cells.In cancer patients, dendritic cell production and function along with other antitumor immune defenses are compromised.Autolo-gous dendritic cells enriched and sensitized in vitro with tumor-associated antigens can effectively elicit host cel-lular immunity against cancer and result in clinical anti-tumor responses through either direct injection or ex vivo generation of antitumor T lymphocytes.In small group studies,clinical response rates have reached50% in patients with advanced stage of cancer.These cellular products caused minimal side effects and were well tolerated.The isolation and preparation of clinical grade dendritic cells have been driven by transfusion medicine specialists who are well versed in similar processes for hematopoietic stem-cell preparation.The purpose of this article is to review the mechanisms of tumor im-mune surveillance and the biology of dendritic cells rel-evant to tumor antigen presentation,sensitization,and T-lymphocyte https://www.wendangku.net/doc/aa8150289.html,rmation on tumor-asso-ciated antigens and clinical trial results with dendritic cell–based cancer immunotherapy are summarized.The potential challenges for blood banking/transfusion medicine involving both technical and regulatory issues are discussed.

?2004Elsevier Inc.All rights reserved.

S URGERY,CHEMOTHERAPY,and radiation therapy either alone or in combination have been the traditional treatments for cancer patients. The modern concept of immunotherapy was intro-duced in the1980s and led to the emergence of cytokine-based therapy.However,cytokines are only part of the potential immunotherapy arsenal, and efforts are in progress to develop cellular-based approaches,designed to activate the host immune system and defeat tumor immune escape. These involve both the ex vivo generation and infusion of activated,clonal antitumor-speci?c lymphocytes,and the in vivo stimulation and ex-pansion of endogenous antitumor-speci?c lympho-cytes by the administration of ex vivo sensitized antigen-presenting cells.The latter is referred to as a cellular-based cancer vaccine.The success of initiating immunologic antitumor surveillance de-pends greatly on appropriate tumor antigen presen-tation.The discovery of dendritic cells(DCs)as potent APC has opened new possibilities for cel-lular-based immunotherapy of cancer.The remark-able ability of DCs to take up,process,and activate naive CD4?and CD8?T lymphocytes makes them ideal candidates for cellular based cancer vaccines as well as for the ex vivo generation of speci?c autologous antitumor T lymphocytes. Promising results have been reported from clinical trials of DC-related cancer immunotherapies.The purpose of this article is to review the biology of DC relevant to antigen presentation and stimula-tion of antitumor immune responses.The potential clinical applications and challenges for transfusion medicine are also discussed.

MECHANISMS OF TUMOR-IMMUNE ESCAPE AND RATIONALE FOR DC-RELATED

IMMUNOTHERAPIES

The interest and optimism in immunotherapy of cancer are based on broadly accepted immunolog-ical principles.Foremost is the existence of tumor-speci?c immune responses in cancer patients.In immune competent individuals,there is an ongoing and vigorous immune surveillance against abnor-mal cells.Abnormal refers to the expression of non-self antigenic determinants capable of eliciting a potent T-cell response.Increased incidence of neoplasms,particularly lymphoproliferative disor-ders,has been seen in posttransplant patients on immunosuppressive therapy.1Conversely,general-ized immune stimulation can trigger antitumor re-sponses.For example,spontaneous tumor regres-sion has been reported in cancer patients after a severe infection.2A similar concept was tested From the Departments of Pathology and Laboratory Medi-cine;and Medicine,University of Wisconsin-Madison,Madi-son,WI.

Address reprint requests to James S.Malter,MD,Depart-ment of Pathology and Laboratory Medicine,Waisman Center, 509T,University of Wisconsin-Madison,1500Highland Ave-nue,Madison,WI53705.E-mail:jsmalter@https://www.wendangku.net/doc/aa8150289.html, ?2004Elsevier Inc.All rights reserved.

0887-7963/04/1803-0005$30.00/0

doi:10.1016/j.tmrv.2004.03.005

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Transfusion Medicine Reviews,Vol18,No3(July),2004:pp189-202

over a century ago,when live bacterial cultures were given to patients with the intent of nonspe-ci?cally activating the immune system against can-cer.3A corollary to the?rst principle is that cancer cells have either abnormal genes or abnormal gene regulation leading to the production of mutant pro-teins and possibly proteins uniquely associated with cancer.In theory,tumor speci?c proteins pro-vide unique targets for an immune response.The ?nal important concept is that HLA molecules,if expressed on the tumor cell surface,will display peptides derived from most intracellular proteins. Therefore,the whole spectrum of abnormal pro-teins,irrespective of their subcellular locations, may be available for immune recognition.Thus, even mutant p53or other aberrant transcriptional control proteins could be presented and ultimately attacked.

With our greater understanding of tumor immu-nology and advances in technology,there has been increasing evidence and enthusiasm for cellular based immunotherapy of cancer.Until relatively recently,standard therapies for hematologic malig-nancies included high-dose chemotherapy fol-lowed by hematopoietic stem cell(HSC)transplant for reconstitution of the bone marrow.However, several clinical studies showed that patients with an allogeneic HSC transplant and graft-versus-host disease had higher rates of long-term survival with less relapse than those who received autologous marrow or marrow from a HLA identical donor.4 This observation suggested that graft-versus-host disease had a positive treatment impact,although with signi?cant side effects.Such experience ra-tionalized the infusion of HSC donor lymphocytes into HSC recipients with relapse.Under certain conditions,complete remission was achieved.5,6 This?nding contributed to the current treatment of relapsing leukemia posttransplant with allogeneic T lymphocytes.There is general consensus that allogeneic HSC transplantation is curative for some malignancies at least in part because of the graft-versus-tumor effects mediated by donor T cells.This shows the power of T-cell mediated antitumor immunity and the importance of the presence and recognition of tumor associated anti-gens(TAAs).The search for TAAs and antitumor-speci?c T lymphocytes is continuing.Our primi-tive understanding of TAAs may explain why some patients who are immune competent fail to “reject”tumor despite the infusion of activated T cells.

The mechanisms of tumor-immune escape are likely multiple and include cancer immuno-edit-ing,7natural selection of tumor variants,8expres-sion of Fas(Apo-1/CD95)ligand to gain immune privilege,9and the secretion of vascular endothelial growth factors that inhibit the functional matura-tion of DCs.10It is important to appreciate that a tumor large enough to be detected clinically has billions of cells,often derived from a single clone.11This“survivor”has multiplied despite tre-mendous selection pressure by host immune sur-veillance.These tumor cell variants often have limited tumor-antigen expression or loss of antigen by mutations or deletions.12Both downregulation of HLA class I expression that reduces antigen presentation as well as the secretion of inhibitory cytokines including interleukin(IL)-10and trans-forming growth factor?have been observed in tumor cells from patients receiving cancer immu-notherapy.13-15In addition,our immune system has a strong predisposition to delete self-reactive T cells to avoid autoimmunity.Therefore,T cells with high-af?nity T-cell receptors(TCRs)that cross-react with self and tumor antigens are likely to be eliminated by the host immune system.16,17 In cancer patients,antigen presentation is often compromised because of reduced DC production and function,which provides a rational for supple-mentation with ex vivo sensitized DC.Immunohis-tological studies showed immature DC resided within breast cancers,whereas mature DCs were located in the tissue surrounding the tumor.18-21 There was minimal recruitment and activation of DCs within renal cell carcinoma,22prostate can-cer,23transitional cell carcinoma,24and mela-noma.25-27This may re?ect the local production of inhibitory cytokines,which include vascular endo-thelial growth factor,IL-6,and macrophage col-ony-stimulating factor.10,28,29These data further support the“tumor immune escape”hypothesis and made DC a strong candidate for cellular-based cancer immunotherapy.

Typically,TAA-loaded DCs are used to activate T lymphocytes in vivo or ex vivo.In both situa-tions,the source materials(tumor,precursors,and T cells)are collected from the patient for in vitro processing.The goal for ex vivo production and manipulation of or T cells is to promote prolifera-tion and maturation of DC for enhanced antigen

190VOSS,ALBERTINI,AND MALTER

processing and presentation and to initiate or aug-ment clonal expansion of speci?c antitumor T cells in an environment without inhibitory in?uences from the host.Furthermore,individually custom-ized cancer immunotherapy is probably essential, given the heterogeneous nature of tumors.Cus-tomization may also reduce unintended immuno-logic side effects of treatment.

DC IDENTIFICATION AND BIOLOGY RELEVANT TO ANTIGEN PRESENTATION

AND T-CELL STIMULATION

The DC is a unique leukocyte characterized by dendritic morphology and high-density expression of human histocompatibility leukocyte antigen class II molecules(HLA-DR).As its name implies, the DC has multiple branch-like projections ex-tending from the cellular surface.DC can be clas-si?ed into myeloid or lymphoid phenotypes based on antigenic expression.30Although all DC are HLA-DR positive and lineage antigen negative (negative for CD3,CD14,CD19,CD56,and other markers of monocytes,lymphocytes,neutrophils, and macrophages),those positive for CD11c and/or CD123are subgrouped as myeloid(CD11c?, CD123low)or lymphoid(CD11c?,CD123hi). The myeloid and lymphoid DC appear to have different functional properties with the former stim-ulating Th1and the later Th2immune responses.31-33 The Th1response enhances in?ammation and cel-lular immunity characterized by secretion of IL-2, interferon(IFN)-?,and IL-12.The Th2response stimulates B cells and antibody production with secretion of IL-4,IL-5,IL-10,and IL-13.DCs derived from CD14?mononuclear cells from hu-man peripheral blood have many of the functional features of myeloid DC.30

DCs are present in virtually every tissue,where they capture antigens and migrate to secondary lymphoid organs to interact with other immune cells.30In nonlymphoid tissues,DCs are in their immature form and characterized by the abundance of intracellular HLA class II molecules.Approxi-mately0.5%to1.5%of the circulating mononu-clear cells are differentiated DCs that have a short life span in culture despite the supplementation with cytokines.34,35As such,DC precursor enrich-ment and in vitro expansion are usually performed. For example,CD34?hematopoietic stem cells separated from peripheral blood,bone marrow,or human umbilical cord blood can differentiate into DCs.DCs can also be obtained through culture of adherent mononuclear cells or immunoselected CD14?monocytes from peripheral blood.Periph-eral blood provides an accessible and convenient source,which can yield up to2?108DCs from a single1-volume cytapheresis procedure followed by in vitro culture in the presence of granulocyte/ macrophage colony-stimulating factor(GM-CSF) and IL-4.36,37These monocyte-derived DCs are predominantly myeloid type and functionally im-mature.Injection of immature DC can induce an-tigen-speci?c inhibition of effector T-cell function in humans.38Therefore,it is very important to ensure DC maturation has occurred during prepa-ration for cancer immunotherapy.Inducers of DC maturation include bacterial-derived products in-cluding lipopolysaccaride,viral double-stranded RNA,various proin?ammatory stimuli,and me-chanical stress.39LPS or tumor necrosis factor?(TNF-?)is most commonly used in culture to induce DC maturation.40High-level expression of surface HLA-DR,CD83,and CD86is associated with successful maturation.41Peripheral blood-de-rived DCs have been used safely in the majority of human clinical trials and have similar functional activities to DCs derived from other tissue sourc-es.30,42

MOLECULAR BIOLOGY OF DC AND GENE MODIFICATION APPROACHES TO IMPROVE ANTIGEN PRESENTATION FOR

T-CELL STIMULATION

The molecular basis of antigen uptake and pro-cessing by DCs are becoming better understood.A diverse range of receptors on the DC membrane are involved in capturing and internalizing anti-gens,which includes the macrophage mannose re-ceptor,DEC-205multilectin receptor,Fc?R,Fc?R (CD32,CD64),and asialoglycoprotein recep-tor.41,43,44,45Endogenous proteins are processed in the proteosome or cytosol,and resultant peptides are transported into the endoplasmic reticulum and assembled onto HLA class I molecules.HLA class II molecules primarily bind to exogenous peptides produced in phagolysosomes.The peptide-loaded HLA molecules are then redistributed from intra-cellular compartments to the plasma membrane.46 This process corresponds to DC development and functional maturation.The immature DCs are ef-?cient in the uptake and processing of antigens, whereas mature DCs are capable of effectively

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DENDRITIC CELL IMMUNOTHERAPY FOR CANCER

presenting antigens to T lymphocytes.During DC maturation,synthesis of HLA molecules is upregu-lated,which facilitates antigen presentation to cy-totoxic lymphocytes and helper T cells.41

The HLA-peptide complex on DCs is recog-nized by the TCR.However,by themselves,HLA peptide-TCR interactions are insuf?cient to trigger T-cell activation.Costimulatory molecules CD80/ CD86and CD40found on the surface of DCs and CD28,ICOS,and signaling lymphocytic activation molecules on the surface of T cells are required for the stimulation of T cells.47Intercellular adhesion molecule-3serves as a costimulatory ligand for leukocyte adhesion glycoprotein LFA-1on DCs.48 Cell membrane processes of DC and chemokine secretion are believed to facilitate an intimate in-teraction between DC and T lymphocytes,such that an immunological synapse,with centrally lo-cated TCR HLA-peptide complex and costimula-tory molecules surrounded by a ring of stabilizing adhesion molecules is formed.49-51The signaling strength is dependent on the amount of peptide-HLA complex on DCs,the number of costimula-tory molecules,and the longevity of the synapse. To maximize antigen presentation,DCs have been engineered to overexpress essential compo-nents of the synapse or cell-activating molecules. These have included the expression or overexpres-sion of TAAs52,53;cytokines including GM-CSF, IL-12,TNF-?,and IFN-?54,55alone or in combi-nation;and costimulatory molecules.56Modi?ed DCs have shown improved antigen presentation or antitumor responses.57,58DNA-encoding B7 (CD80)has been introduced into melanoma and osteosarcoma to enhance their antigenicity.59,60 The transfected tumor cells were readily recog-nized and destroyed by reactive T cells.Impor-tantly,these reactive T cells were also protective against parental B7-negative tumor cells,59which suggested that only a portion of the tumor cells with transgenic expression of B7was suf?cient to induce an antitumor response to the entire tumor mass.

THE CHOICE OF TUMOR ANTIGENS AND

SENSITIZATION OF DCs

To initiate speci?c antitumor T-lymphocyte re-sponses,appropriate tumor antigens must be iden-ti?ed.Genomic and proteomic technology have enabled large-scale screening of protein expression patterns in various malignancies.Studies have shown that malignant cells express altered normal proteins or,more importantly,proteins uniquely associated with their phenotype.These proteins provide possible antigens to elicit a speci?c and unique antitumor T-cell immune response.61Ex-amples of identi?ed human TAAs recognized by cytotoxic T cells are summarized in Table1ac-cording to their origin or derivation.For practical purposes,a classi?cation scheme based on their potential usefulness for cancer immunotherapy has been developed.62This system made distinctions be-tween unique and shared tumor antigens,which were classi?ed under the general term TAA.An updated list of TAAs recognized by T lymphocytes is avail-able in the peptide database(www.cancerimmunity. org/peptidedatabase/tcellepitopes.htm)with de-tailed information including GeneCard links for the encoding gene and/or the parent protein,anti-Table1.Examples of Identi?ed Tumor-Associated Antigens Recognized by Cytotoxic T Lymphocytes

Viral derived tumor antigens

HPV E6and E7proteins in cervical carcinoma

EBV LMP-1and EBNA-1proteins in Hodgkin’s disease and nasopharyngeal carcinoma

Proteins encoded by point mutated genes or chromosomal rearrangements

ras in carcinomas of lung,colon,pancreas,and many

leukemias

BCR/ABL in chronic myeloid leukemia and acute

lymphoblastic leukemia

PML/RAR-?in acute promyelocytic leukemia

ret in multiple endocrine neoplasia2A and B,familiar

medullary thyroid carcinoma

myc in Burkitt lymphoma

Normal gene products selectively expressed or

overexpressed in the tumor

Telomerase in85%of tumors

Her-2/neu in breast and ovarian cancer

Normal gene products that are tissue-speci?c and expressed by the tumor

Tyrosinase,melano A,gp100,TRP-1,TRP-2in melanoma PSA in prostatic carcinoma

Normal gene products not expressed or minimally expressed in adult tissues

MAGE,BAGE,GAGE,RAGE,and NY/ESO proteins in

melanoma and other tumors

Oncofetal antigens

AFP in hepatocellular carcinoma

CEA in colorectal and pancreatic carcinoma

Differentiation antigens

CD10in B cell leukemia and lymphomas

Idiotypic proteins

Immunoglobulin molecules in B-cell lymphoma)

Minor HLA antigens expressed selectively by hematopoietic cells in allogeneic HSC recipients

192VOSS,ALBERTINI,AND MALTER

gen-expression pattern,peptide antigen sequence, chromosomal location,and mode of T-cell stimu-lation.

Theoretically,the ideal target antigens for can-cer immunotherapy are derived from proteins that are present only on tumor cells and not expressed on normal cells.These antigens must be capable of stimulating cytotoxic T lymphocytes.Candidate antigens include viral gene products in virus-asso-ciated malignancies and proteins encoded by mu-tated endogenous genes,such as bcr/abl and pml/ rar-?fusion proteins,K-ras,and N-ras.63,64Some of the mutations are unique to the tumor of an individual or restricted to very few patients.Be-cause these unique tumor antigens are not shared by the same histological tumors from other pa-tients,their value as an antigenic peptide for cancer immunotherapy in the general population is lim-ited.However,these unique tumor antigens are strictly tumor speci?c and may play an important role in the natural antitumor immune responses and in customized cancer immunotherapy for individ-ual patients.

The so-called shared tumor antigens are a subset of TAAs expressed on many independent tumors, which can be subdivided into shared tumor-spe-ci?c antigens,differentiation antigens,and overex-pressed tumor antigens.The shared tumor-speci?c antigens are of great interest because these proteins are not present in normal adult somatic tissues but are expressed by various tumor types.Testis is the only tissue in which such proteins are normally expressed.However,they cannot be presented and subsequently attacked because male germ cells do not express cell-surface HLA molecules.Thus, these proteins can be considered as tumor speci?c. Prototype antigens of this group are those encoded by the MAGE gene family,which includes MAGE-1,MAGE-3,BAGE,GAGE-1,GAGE-2, and NY-ESO-1.This group of genes frequently maps to the X chromosome,and their regulation is disrupted in malignant cells,including melanoma, sarcoma,non–small-cell lung cancers,head and neck tumors,and transitional cell carcinoma.65 These shared tumor-speci?c antigens are important targets for the development of antigen-speci?c cy-totoxic T cells for cancer immunotherapy. Another group of shared tumor antigens is the differentiation antigens that are expressed in tu-mors as well as in the normal cells from which the tumor has originated.These antigens have been best characterized in melanoma,although they are being actively investigated in many tumors includ-ing prostate cancer.The differentiation antigens can stimulate in vitro cellular immune responses. Both HLA class I–restricted CD8?immunodomi-nant epitopes and HLA class II–restricted CD4?immunodominant epitopes have been character-ized.This group comprises antigens encoded by Melan-A/MART-1,gp100/Pmel17,gp75,65pros-tate-speci?c antigen,and tyrosinase.Tumor differ-entiation antigens are not tumor speci?c,and their use as cancer immunotherapy targets may cause autoimmunity against the corresponding normal tissue(eg,pigmented cells in skin and eyes when MART-1and gp100are used).

The third group of shared tumor antigens is present in a wide variety of normal tissues but overexpressed in many types of tumors(eg,telom-erase,Her-2/neu,MUC1,and p53).Because ubiq-uitous and low-level expression of these genes occurs in normal tissues,utilization of overex-pressed tumor antigens as cancer immunotherapy targets must be carefully considered.The current suggestion is that overexpressed“self-antigens”can be useful if the autoimmune consequences of such treatment are tolerable or locally controllable or if the damaged organ is super?uous or can be readily replaced.66

The TAA classi?cation described earlier is arbi-trary.Some proteins are dif?cult to?t exactly into a speci?c group.For example,mucins are a type of proteins that can give rise to unique tumor anti-gens.In some pancreatic,ovarian,and breast car-cinomas,peptides derived from abnormally glyco-sylated cell-surface mucins can be recognized by cytotoxic T cells.However,the mucin-derived MUC1peptide(STAPPVHNV)is present in nor-mal glandular epithelium and is considered as an overexpressed shared antigen.61,67

DC can be sensitized in vitro by exposure to either crude tumor antigens(eg,whole tumor cell lysates or speci?cally de?ned tumor antigens if known and available).Whole-cell vaccines pre-pared by the fusion of DC and tumor cells can initiate effective antitumor responses.68-70Crude tumor antigens derived from whole-cell lysates are simple to prepare and contain the entire spectrum of TAAs,even those not fully characterized(eg, unique antigens from individual tumors).Theoret-ically,the simultaneous sensitization to multiple distinct protein antigens may minimize mutation-

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DENDRITIC CELL IMMUNOTHERAPY FOR CANCER

driven tumor immune escape.Utilization of whole tumor cell antigens derived from apoptotic or irra-diated cells is also being evaluated and compared with lysates of viable tumor cells for their ef?cacy in sensitizing DC and stimulating T-cell responses. Studies in animal models showed that tumor cell lysates have equal or greater potency than apopto-tic or irradiated tumor cells in both immune priming and antitumor response.71,72However,low-fre-quency antigens may be inadequately represented in such preparation with correspondingly reduced DC sensitization and T-cell activation.

After proteins derived from malignant cells are endocytosed and processed in DCs,the resultant peptides are presented by HLA molecules.The composition of presented peptides varies based on the genetic and biochemical proclivities of an in-dividual’s DC and tumor antigenic characteristics. When tumor antigen peptides have been identi?ed, they can be used for DC sensitization.This is a more de?ned system permitting peptides to be pro-duced using good manufacturing practices(GMPs) and T-cell responses monitored by peptide-HLA tetramer staining.However,isolated peptide used without the context and orientation of the original protein may fail to fully activate T cells.Other drawbacks for using single isolated TAA peptide include increased chance for mutation-driven tu-mor immune escape and reduced ef?cacy caused by short half-life.73,74Modi?ed TAA peptides have been constructed that showed increased T-cell stimulatory activity.75For example,a carcinoem-bryonic antigen(CEA)-derived peptide(amino acids605-613)has been modi?ed by substituting as-partate for asparagine at position610,which re-sulted in increased T-cell responses against CEA in vitro.76Peptide antigenicity has also been im-proved by conjugation to the hepatitis B core pro-tein to form a virus-like particle.77Multiple pep-tides or multiple copies of the peptide can be linked to a carrier,such as keyhole limpet hemo-cyanin or?-galactosidase to prolong the half-life.77 Other strategies include using specially synthe-sized retro-inverso peptides;pseudopeptides with amide bond surrogates77;combinations of peptides capable of stimulating both cytotoxic and helper T cells,and improved delivery through receptors, liposomes,or bacterial toxin-peptide fusions.39,78 Reverse-transcriptionpolymerase chain reaction is useful to identify the expression of particular pep-tides in individual tumors for accurate targeting,79although controls for normal cell contamination must be included.In general,using generic peptides as the source of tumor antigen may not cover mutated epitopes peculiar to the patient’s own tumor.80 HUMAN CLINICAL TRIALS WITH

DC-RELATED IMMUNOTHERAPIES

OF CANCER

Human clinical trials(HCTs)using cancer vac-cines with tumor antigen-loaded DCs or the infu-sion of ex vivo–generated antitumor T cells stim-ulated by TAA-loaded DCs are ongoing.The goals have been either shrinkage of existing tumor,usu-ally in patients with advanced disease,or prophy-lactic therapy.68Applications in a wide variety of malignancies have been evaluated,including mel-anoma,advanced renal cell carcinoma,metastatic adenocarcinoma of breast and pancreas,prostatic carcinoma with metastasis,non–small-cell lung carcinoma,ovarian carcinoma,hepatocellular car-cinoma,and hematological malignancies.Repre-sentative clinical trial results are summarized in Table2.The majority of the studies are in phase I or II.The feasibility and safety of cellular-based immunotherapy have been repeatedly shown.83 In vivo cellular-immune responses speci?cally against target tumor antigens were detected by delayed-type hypersensitivity(DTH)and cytokine production in patients received TAA-loaded DC vaccines.The demonstration of in vivo immuno-genicity from these different vaccinations has pro-vided leads for ongoing clinical development.It is encouraging that some evidence of antitumor ac-tivity has been achieved in these early-to mid-phase clinical trials.80,87

Vaccination using intranodal injection of autol-ogous tumor lysate-pulsed DC was performed on 10patients with cutaneous T-cell lymphoma who were refractory to standard treatment.Tumor-spe-ci?c DTH developed in all vaccinated patients,and 5of10patients had objective clinical responses, including4of10partial remission and1of10 complete remission.The mean partial remission duration was10.5months,whereas the complete remission has lasted more than19months.91In the HCT conducted by O’Rourke et al,80patients with American Joint Cancer Committee stage IV mela-noma were vaccinated with DC sensitized by irra-diated tumor cells at ratio of4DCs to1tumor cell.

A complete priming phase of6cycles of either

0.92?106or5?106DCs were given to12

194VOSS,ALBERTINI,AND MALTER

patients by intradermal injection at 2-week inter-vals.Durable complete responses lasting an aver-age duration of more than 35months were seen in 3of 12patients,and partial responses were ob-served in 3of 12patients according to World Health Organization criteria.No signi ?cant side effects have been reported in this study.80

Considering the advanced stages of these dis-eases and current lack of alternative treatment op-tions for the majority of them,positive results in these HCTs are exciting.However,optimal dose delivery and immunization schedules are empiric.Immunologic monitoring methods predictive of clinical outcome need to be improved.Speci ?cally,rapid detection of in vivo antitumor-speci ?c immune response remains problematic.In vitro analysis of antigen-speci ?c cytotoxic T cells is currently available and includes target killing,cy-tokine production,CDR3polymorphism analysis,and tetramer staining.92,93A recent consensus con-ference held by the Society for Biologic Therapy 94suggested (1)future vaccine studies should corre-late T-cell function with clinical outcome;(2)quantitative tetramer-based assays should be com-pared with functional assays,such as enzyme-linked immunospot or ?ow cytometry analysis for cytokine production;and (3)proliferation assays are imprecise and should be de-emphasized.Mo-lecular cytokine analysis by reverse-transcription polymerase chain reaction is promising but needs validation as a surrogate for vaccine potency and clinical effect.94In summary,current results sup-port the use of a functional assay (eg,the enzyme-linked immunospot or cytokine ?ow cytometry in combination with a quantitative assay for immune monitoring).

Table 2.Representative Clinical Trials Using Dendritic Cell Related Cancer Immunotherapy

Authors/Phase of Studies

Type of Cancer

Target Antigens

Results

Adverse Effects

O ’Rourke MG et al 80

phase I/II

Stage IV melanoma Irradiated tumor cells Clinical:3/12CR*13/12PR*2,6/12PD*3

Minimal Marten A et al 81

phase I/II

Renal cell carcinoma Whole tumor cell lysate DTH:3/13Clinical:1/15PR,7/15SD*4,7/15PD

No Iwashita Y et al 82

phase I

Unresectable primary liver cancer

Whole tumor cell lysate DTH §:7/10AFP decrease:2/10Clinical:1/10PR No Pecher G et al 83

phase I/II Advanced pancreatic,breast,and papilla of Vater adenocarcinoma MUCI

DTH:3/10IFN-?secretion:4/10Clinical:1/10SD,9/10PD

No

Pinilla J et al 84

phase II Chronic myeloid leukemia

Bcr/abl peptide

DTH:14/14IFN-?secretion:11/14in CD4?,4/14in CD8?

No

Su Z et al 85

phase I Metastatic renal cell carcinoma

Tumor RNA Expansion of tumor-speci ?c T cells:6/7

No Lin CL et al 86

Nasopharyngeal carcinoma EBV peptide

T cell proliferation:9/16Clinical:2/16PR

Minimal Timmerman JM et al 87

phase I/II B-cell lymphoma

Tumor speci ?c Immunoglobulin

T cell proliferation:8/10Clinical:2/10CR,2/10PR,Minimal Stift A et al 88

phase I/II

Pancreatic,hepatocellular,cholangiocellular,and medullary thyroid carcinoma

Whole tumor cell lysate

DTH:18/20Objective

changes in tumor size and tumor markers:7/20No

Geiger ID et al 89

phase I Pediatric solid tumors:

Neuroblastoma,sarcoma,renal tumors Tumor cell lysate and KLH #DTH:7/10IFN-?secretion:6/10Clinical:1/10PR,5/10SD

No

Disis M et al 90

phase I Stage III/IV breast

carcinoma,ovarian cancer in remission

HER-2/neu peptide and Flt3ligand DTH:3/8IFN-?secretion:10/10

Minimal

Maier T et al 91

Cutaneous T-cell lymphoma

Tumor lysate and KLH

DTH:8/8IFN-?production:3/5Clinical:4/10PR,1/10CR

Minimal

Abbreviations:CR*1,complete response;PR*2,partial response;PD*3,progressive disease;SD*4,stable disease;DTH §,delayed-type hypersensitivity;KLH,immunogenic carrier protein keyhole limpet hemocyanin.

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DENDRITIC CELL IMMUNOTHERAPY FOR CANCER

In addition to in vivo and ex vivo DC-induced stimulation and expansion of cytotoxic T cells, other strategies have also been tested in clinical trials.Instead of DC precursor collection and sen-sitization by TAA in vitro,irradiated autologous tumor cells were admixed with Calmette-Gue′rin Bacillus and injected intradermally at2sites ap-proximately10cm below the inguinal crease in both thighs.95After7days,lymphocytes were iso-lated from vaccine-primed lymph nodes,second-arily activated with anti-CD3monoclonal anti-body,and expanded in the culture medium supplemented with IL-2.The activated autologous lymphocytes were infused intravenously along with bolus https://www.wendangku.net/doc/aa8150289.html,plete responses(4/34)and partial responses(5/34)were reported in patients with stage IV renal cell carcinoma for an overall response rate of27%with no signi?cant side ef-fects.95Another variation was to isolate antimela-noma-speci?c T lymphocytes from total tumor-in?ltrating T cells(TILs)followed by clonal expansion in vitro.TILs were obtained from re-sected melanomas from13patients with advanced stage disease and screened for cytokine secretion after exposure to autologous tumor cells.Tumor reactive T cells were expanded in the culture with irradiated allogeneic feeder cells,anti-CD3anti-body,and IL-2.Reinfusion of these selected T-cell clones to patients after nonmyeloablative chemo-therapy resulted in persistent clonal T-cell repopu-lation and proliferation in vivo.More than50% reduction in tumor size including metastatic le-sions in lungs and liver was observed in6of13 patients.An18-year-old patient with extensive metastatic melanoma remained disease free for2 years after the treatment.Side effects included vitiligo in4of13patients and bilateral acute anterior uveitis in1of13patients that was relieved by administration of steroid eye drops.66The im-pressive results have drawn attention from the sci-enti?c?eld as well as from the public.96

PREPARATION OF CLINICAL GRADE

PRODUCTS FOR DC-RELATED

IMMUNOTHERAPY

There is an increasing need for the development of practical,reliable,and ef?cient methods to pro-duce large numbers of clinical grade DC.The production of DC for clinical applications must comply with good tissue practice and GMP guide-lines.39Rouard et al37has developed a closed and single-use system using elutriation with an aphere-sis device.Approximately1.8to9.3?108CD14?monocytes were harvested in less than an hour with70%to90%purity by this modi?ed leuka-pheresis procedure.A blood prime to maintain hemodynamic stability is required in children less than20kg.89Administration of mobilizing cyto-kine or other adjuvant before the procedure is not necessary.In patients with metastatic carcinomas or hematological malignancies,tumor cells can be present in the peripheral blood.In this case,the collected DC precursors may be contaminated by tumor cells.Consensus has yet to emerge over the need to remove tumor cells for this therapeutic modality.Positive or negative immunological se-lection techniques are available for further DC puri?cation or tumor cell depletion if proven to be necessary.

From isolated CD14?DC precursors,func-tional DC can be generated by a6-day culture with GM-CSF,IL-4,and further matured in the pres-ence of TNF-?or a cocktail of IL-1?,IL-6, TNF-?,and prostaglandin-E2with an average yield rate from20%to30%.36,97Optimal culture condi-tions need to be further de?ned,especially related to the type and concentration of serum supple-ments,cell purity,and cell density.98Subtle changes in culture conditions,such as different pathogens,cytokines,cocultured cell populations, or antigens,as well as the origin of the serum supplement(fetal calf serum versus human AB serum versus autologous plasma)can alter DC gene expression and function.99This interesting phenomenon is just another re?ection of the diver-sity and complexity of DC as an integrating and regulatory center for immune responses.30,100 Therefore,the in vitro conditions must be standard-ized and the outcome carefully monitored.98Cur-rently,X-VIVO15medium(Cambrex,East Ruth-erford,NJ),AIM-V serum-free medium(Gibco, BRL,Gaithersburg,MD),and RPMI-1640(Gibco, BRL)supplemented with10%allogeneic human AB serum or autologous serum have been used in cell cultures for different clinical trials.Only phar-maceutical grade cytokines can be used.The ex vivo–generated DC can be cryopreserved and re-tain82%functional viability up to24months after ?80°C freezing and storage using extracellular hydroxyethyl starch or intracellular dimethylsulf-oxide reagents.101DC precursors and lymphocytes can be cryopreserved in the same fashion.Thawed

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CD14?mononuclear cells maintained92%viabil-ity and were able to differentiate into immature and mature DC.

Sensitization of DC with tumor antigens is the next step in DC-based cancer vaccine preparation.

A standard tumor procurement protocol has been used with minimal modi?cations in published clin-ical trials.In general,tumors are obtained by sur-gical resection.After con?rmation of the diagnosis on frozen section,the sterile specimen is dissoci-ated by combinations of mechanical and enzymatic steps to create a single-cell suspension of tumor cells for culture or cryopreservation.70Irrespective of the tumor type,normal cells including?bro-blasts,endothelial cells,and lymphocytes are also present in cell suspension prepared in this fashion. Theoretically,a pure tumor cell preparation is ideal to maximize the ef?cacy of a DC vaccine and reduce the incidence of an autoimmune response. However,HCTs have shown minimal autoimmu-nity with impure antigen preparations.88,89Should greater purity be necessary,immunoselection or ?ne-needle aspiration with subsequent limiting di-lution can be performed to isolate individual tumor cell for in vitro expansion.The optimal cell-to-antigen ratios for DC sensitization remain to be established.This will likely vary depending on the type and source of antigens.With whole tumor cell lysates,the currently recommended DC to tumor cell equivalent ratio is1:1to2.5:189;with irradi-ated whole tumor cells,the ratio is4to1.80 After antigen uptake,further DC maturation is mandatory.The infusion of immature but antigen-loaded DC suppressed antitumor immunity in some recipients.38Fully mature DC show high-level expression of surface HLA class II,CD86, and CD83that can be determined by?ow cytom-etry analysis.41The tumor antigen-loaded mature DCs are then injected as a DC vaccine or used as a stimulator for ex vivo generation of antitumor T lymphocytes.According to the current protocols for DC-based cancer vaccines,1to10?106 antigen-loaded DC can be injected subcutaneously near lymph nodes or directly into lymph nodes under ultrasound guidance at weekly or monthly intervals.80The administered dose and treatment intervals have been variable in different HCTs. Studies to systematically evaluate dosing regimen with clinical outcome are needed.

Autologous T lymphocytes from peripheral blood or TIL cells can be stimulated by TAA 8loaded DC to initiate or augment antitumor re-sponses.A1:5to1:10ratio of DC to lymphocytes is usually adopted for co-culture.The stimulated lymphocytes are then given by intravenous infu-sion.The number of lymphocytes transfused has ranged from2.3to13.7?1010.66Lymphocyte preparations stimulated by impure tumor antigens will contain a mixture of T cells reactive to tumor associated or nonassociated antigenic epitopes. Present techniques for the isolation of speci?c an-titumor T cells by clonal selection through limiting dilution and screening for tumor or tumor antigen-speci?c cytokine production is time consuming, labor intensive,and clinically impractical.Tet-ramer assay by?ow cytometry can identify the presence of high-af?nity cell surface HLA mole-cules provided the antigen peptide sequence is known.This assay does not de?ne antitumor func-tionality,however.Therefore,more ef?cient and timely selection of speci?c antitumor T cells for cancer immunotherapy is sorely needed.The ro-setting of T cells to tumor cells for rapid enrich-ment of speci?c antitumor T lymphocytes is cur-rently under investigation in our laboratory.102

In addition to the technical aspects of DC col-lection,processing,storage,and administration discussed previously,appropriate donor selection, bene?t/risk evaluation,and informed consent must be addressed by both clinicians and transfusion medicine specialists.Patients with multiple my-eloma,lymphoma,and metastatic carcinoma can function as donors for autologous DC collec-tion.87,103However,patients with human immuno-de?ciency virus infection and clinically signi?cant autoimmune disease on immunosuppressive ther-apy should be excluded because of impaired cel-lular immunity.Pretreatment DTH responses against Mumps,Candida,and Trichophytin are helpful predictors of patient’s immunization poten-tial.79The patient/donor selection should also con-sider the immunogenicity of the individual tumor, which varies depending on the type and expression level of tumor antigens in the context of HLA class I,as well as the expression of the adhesion mole-cules and costimulatory factors on the tumor cells. These properties can be evaluated by immunologic and molecular techniques.For example,immuno-logical screening for HLA class I antigen expres-sion on malignant cells can help determine if the patient is a suitable candidate for T-cell–based immunotherapy because cytotoxic T cells recog-

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nize TAA under the context of HLA class I mol-ecules.104Cancer patients have shown variable re-sponses to the same approach of immunotherapy regardless of identical morphological diagnosis in the same clinical stage,66which may re?ect the unique immunogenicity of the individual tu-mor.62,105-107The current tumor grading system based on histological features of the neoplasm may not provide suf?cient information to guide cancer immunotherapy.Clearly,far greater understanding of the immunology and biology of individual tu-mors is necessary to improve patient selection, prognosis,and clinical decision making.The rap-idly developing technology of gene chips and pro-teomic analysis is rede?ning the expression pro-?les of tumor with low or high immunogenicity. The experience with allogenic granulocyte or lymphocyte transfusions can be applied to cancer immunotherapy with autologous DC or T lympho-cytes.The risks for signi?cant transfusion reac-tions certainly exist.The extensive in vitro manip-ulations can introduce chemicals,foreign proteins, cytokines,shed antigens,and other exogenous ma-terials into the product.The possibility of provok-ing an autoimmune reaction is a special concern. Results from phase I/II clinical trials have,how-ever,shown minimal side effects.Even through autoantibodies(antinuclear antibodies,anti–Sjo¨g-rens Syndrome antigen,and anti–double-stranded DNA)were detected serologically in some recipi-ents,clinical autoimmune disease was rare and reversible by treatment.90Uveitis has happened occasionally in patients receiving T-cell infusion targeted at tumor differentiation antigens MART-1/Melan-A,which was controlled by steroid eye drops.66

Cost-effectiveness is a signi?cant concern.108 The potential areas to be modi?ed for reducing cost,improving feasibility,and clinical effective-ness include(1)eliminating in vitro manipulation of T cells by direct TAA-loaded DC vaccination and(2)eliminating the DC collection and culture steps by either direct injection of irradiated tumor cells with Calmette-Gue′rin Bacillus followed by T-cell collection from primed lymph nodes94or isolation of speci?c antitumor T cells from TIL.66 There are concerns with applying peptide alone directly into subcutaneous tissue without being loaded onto DC because this approach can spe-ci?cally tolerize T cells and enhance tumor growth.109Current clinical response rates,albeit in small group studies,have reached27%to50% with signi?cantly extended survival for up to2to 3years.66,80,94These results were achieved in the patients with end-stage disease who have failed other cancer treatments.As with other therapeutic interventions,early detection and treatment based on patients’immunotherapeutic potential and tu-mor immunogenicity are very likely to signi?-cantly improve the clinical outcome and overall cost-effectiveness of cellular based immunother-apy of cancer.

QUALITY ASSURANCE/QUALITY

CONTROL ISSUES Harvesting,preparation,preservation,distribu-tion,and administration of human hematopoietic stem cells(HSC)from peripheral blood,bone mar-row,and umbilical cord blood has been in clinical practice for many years.The principle of quality assurance and quality control for HSC processing should be applied to DC preparation.DC process-ing requires more extensive in vitor manipulation than HSC.Efforts have been made to create a uniform,closed DC culture system.40The Ameri-can Association of Blood Banks will release stan-dards for somatic cell laboratories with a projected implementation date of June1,2004.A single cellular therapy standards program unit will emerge from existing standards for Hematopoietic Progenitor and Cellular Product Services and Cord Blood and will be applicable to DC processing as well.110

Despite signi?cant optimism,many obstacles re-main.By its nature,each cellular-based immuno-therapy product is unique and will be manufac-tured for the treatment of a single individual.It will be time consuming,labor intensive,and expensive. Personnel training,patient-speci?c labeling,prod-uct segregation and veri?cation,and contamination prevention will be the focus of quality assurance. Specimen labeling,documentation,and tracking are essential.For products of cellular-based immu-notherapy,in-process controls,including charac-terization and functional analysis of cellular mate-rial,determination of cell number and viability, and testing for adventitious agents,will be oblig-atory.In addition,physiochemical features,includ-ing pH,storage temperature,and viscosity or cell aggregation of the product need to be assessed before the product can be released.111There are no generally available standard operating procedures

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at the present time.Well-planned clinical trials with careful and systematic data analysis will pro-vide valuable information.

A process similar to the manufacture of infec-tious disease vaccines may evolve.Licensed bio-logical products,including vaccines,must be safe, pure,potent,and manufactured consistently ac-cording to the current GMPs.Based on US Food and Drug Administration guidelines,the de?nition of safe is relatively free from harmful effect when prudently administered;pure is de?ned as rela-tively free from extraneous matter.The common principles for vaccine production and quality con-trol include the following:detailed manufacturing procedures to ensure consistency of production, de?ned compatible components,product character-ization and speci?cations,adventitious agent test-ing,examination of extraneous materials,and sta-bility of the product including genetic stability.

CONCLUSIONS

The feasibility,safety,and immunogenicity of DC-related immunotherapy for cancer have been shown in multiple clinical trials that included pa-tients with a variety of malignancies and in various clinical conditions,especially those with end-stage disease.Clinical antitumor effects including dura-ble complete responses have been achieved in small group studies.The promising results,mini-mal side effects,and improved quality of life for cancer patients make this treatment modality ex-tremely attractive when compared with conventional chemotherapy.Effective therapeutic development and implementation require a multidisciplinary collaboration.More phase III and larger-scale clin-ical trials will facilitate the development of good laboratory practices and good clinical practices, speci?cally the hospital-based manufacturing plat-form for therapeutic cellular products,optimized patient selection,dosing regimen,and monitoring parameters.From a scienti?c point of view,studies are needed to further identify and characterize TAAs and other key factors that contribute to consistent and persistent activation of antitumor T cells and increased clinical effectiveness.There will be a constant struggle between the host im-mune system and the tumor because the outgrowth of escape variants can occur during treatment.The addition of DC-based immunotherapy to conven-tional treatments is likely to promote host antitu-mor immunity and bring substantial clinical bene-?ts to more patients with cancer.

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202VOSS,ALBERTINI,AND MALTER

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六年级美术下册--《追寻文明的足迹》说课教案(人美版)

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奇琴伊查库库尔坎金字塔英文介绍

The stepped pyramids, temples, columned arcades, and other stone structures of ChichénItzá were sacred to the Maya and a sophisticated urban center of their empire from A.D. 750 to 1200. Viewed as a whole, the incredible complex reveals much about the Maya and Toltec vision of the universe—which was intimately tied to what was visible in the dark night skies of the Yucatán Peninsula. The most recognizable structure here is the Temple of Kukulkan, also known as El Castillo. This glorious step pyramid demonstrates the accuracy and importance of Maya astronomy—and the heavy influence of the Toltecs, who invaded around 1000 and precipitated a merger of the two cultural traditions. The temple has 365 steps—one for each day of the year. Each of the temple’s four sides has 91 steps, and the top platform makes the 365th. Devising a 365-day calendar was just one feat of Maya science. Incredibly, twice a year on the spring and autumn equinoxes, a shadow falls on the pyramid in the shape of a serpent. As the sun sets, this shadowy snake descends the steps to eventually join a

库库尔坎金字塔

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下端，有一个带羽行的大蛇头石刻，蛇头高1.43米，长达1.80米，宽1.07米，蛇嘴里吐出一条大舌头，颇为独特。这座古老的建筑，在建造之前，经过了精心的几何设计，它所表达出的精确度和玄妙而充满戏剧性的效果，令后人叹为观止：每年春分和秋分两天的日落时分，北面一组台阶的边墙会在阳光照射下形成弯弯曲曲的七段等腰三角形，连同底部雕刻的蛇头，宛若一条巨蛇从塔顶向大地游动，象征着羽蛇神在春分时苏醒，爬出庙宇。每一次，这个幻像持续整整3 小时22 分，分秒不差。这个神秘景观被称为“光影蛇形”。每当“库库尔坎”金字塔出同蛇影奇观的时候，古代玛雅人就欢聚在一起，高歌起舞，庆祝这位羽毛蛇神的降临。库库尔坎金字塔，是玛雅人对其掌握的建筑几何知识的绝妙展示，而金字塔旁边的天文台，更是把这种高超的几何和天文知识表现得淋漓尽致。[1] 建造工艺 库库尔坎金字塔资料图(5张) 在没有金属工具、没有大牲畜和轮车的情况下，古代玛雅人却能够开采大量重达数十吨的石头，跋山涉水、一路艰辛地运到目的地，建成一个个雄伟的金字塔。金字塔最高的可达70米，其规模之巨大、施工难度之高，令人吃惊。古代玛雅的金字塔和古埃及的金字塔在建筑形式上有着明显的不同。埃及的金字塔的塔顶是尖的，而玛雅金字塔却是平顶，塔体呈方形，底大顶小，层层叠叠，塔顶的台上还建有庙宇；在用途上也不一样：埃及金字塔是法老的陵墓，而玛雅的金字塔除个别外，一般是用来祭祀或观察天象的。除金字塔外，玛雅人还兴建了不少功能性强，技艺高的民用建筑，主要有：房屋（包括庙宇、府邸、民居等）、公共设施（球场、广场、集市等）、基础设施（桥梁、大道、码头、堤坝、护墙等）和水利工程（水渠、水库、水井、梯田）等。 历史溯源 玛雅文化的历史

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